Et700 75m

Single molecule imaging experiments were performed at room temperature (23 °C) using an inverted Nikon Ti2 microscope using a 100 x oil immersion Nikon TIRF objective (1.49 NA). The x-axis and y-axis positions were controlled using a Nikon motorized stage, joystick, and Nikon’s NIS element software. Fluorescently labeled proteins were excited with one of three diode lasers: a 488 nm, a 561 nm, or 642 nm (OBIS laser diode, Coherent Inc, Santa Clara, CA). The lasers were controlled with a Vortran laser launch and acousto-optic tuneable filters (AOTF) control. Excitation and emission light was transmitted through a multi-bandpass quad filter cube (C-TIRF ULTRA HI S/N QUAD 405/488/561/642; Semrock) containing a dichroic mirror. The laser power measured through the objective for single particle visualized was 1–3 mW. Fluorescence emission was captured on an iXon Life 897 EMCCD camera (Andor Technology Ltd., UK) after passing through one of the following 25 mm a Nikon Ti2 emission filters mounted in a Nikon emission filter wheel: ET525/50M, ET600/50M, and ET700/75M (Semrock).

Membrane binding and lipid dephosphorylation reactions reconstituted on SLBs were visualized using an inverted Nikon Eclipse Ti2 microscope using a 100× Nikon (1.49 numerical aperture) oil immersion TIRF objective. TIRF-M images of SLBs were acquired using an iXion Life 897 EMCCD camera (Andor Technology Ltd). Fluorescently labeled proteins were excited with either a 488 nm, 561 nm, or 637 nm diode laser (OBIS laser diode; Coherent, Inc) controlled with a Vortran laser drive with acousto-optic tunable filters control. The power output measured through the objective for single-particle imaging was 1 to 2 mW. Excitation light was passed through the following dichroic filter cubes before illuminating the sample: (1) ZT488/647rpc and (2) ZT561rdc (ET575LP) (Semrock). Fluorescence emission was detected on the iXion Life 897 EMCCD camera positioned after a Nikon emission filter wheel housing the following emission filters: ET525/50M, ET600/50M, and ET700/75M (Semrock). All experiments were performed at room temperature (23 °C) for both biochemistry and live-cell imaging. Microscope hardware was controlled by Nikon NIS elements.